Practical Application of Rodent Transporter Knockout Models to Assess
Brain Penetration in Drug Discovery

Eneberg, Elin; Jones, Christopher C.; Jensen, Tommy R.; Langthaler, Kristine; Bundgaard, Christoffer

doi:10.2174/1872312815666220222091032

Cited by 4 publications

(3 citation statements)

References 42 publications

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“…Terminal blood samples were collected from all animals. Brain samples were prepared by homogenization using ultrasonication as described previously [ 39 ] and analyzed together with the plasma samples using LC–MS/MS [ 39 ]. The lower limit of quantification was determined to 1.0 ng/mL in plasma and 5 ng/g in brain tissue for both eletriptan and diphenhydramine.…”

Section: Methodsmentioning

confidence: 99%

The putative proton-coupled organic cation antiporter is involved in uptake of triptans into human brain capillary endothelial cells

Svane,

Pedersen,

Rodenberg

et al. 2024

Fluids Barriers CNS

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Background Triptans are anti-migraine drugs with a potential central site of action. However, it is not known to what extent triptans cross the blood–brain barrier (BBB). The aim of this study was therefore to determine if triptans pass the brain capillary endothelium and investigate the possible underlying mechanisms with focus on the involvement of the putative proton-coupled organic cation (H+/OC) antiporter. Additionally, we evaluated whether triptans interacted with the efflux transporter, P-glycoprotein (P-gp). Methods We investigated the cellular uptake characteristics of the prototypical H+/OC antiporter substrates, pyrilamine and oxycodone, and seven different triptans in the human brain microvascular endothelial cell line, hCMEC/D3. Triptan interactions with P-gp were studied using the IPEC-J2 MDR1 cell line. Lastly, in vivo neuropharmacokinetic assessment of the unbound brain-to-plasma disposition of eletriptan was conducted in wild type and mdr1a/1b knockout mice. Results We demonstrated that most triptans were able to inhibit uptake of the H+/OC antiporter substrate, pyrilamine, with eletriptan emerging as the strongest inhibitor. Eletriptan, almotriptan, and sumatriptan exhibited a pH-dependent uptake into hCMEC/D3 cells. Eletriptan demonstrated saturable uptake kinetics with an apparent Km of 89 ± 38 µM and a Jmax of 2.2 ± 0.7 nmol·min−1·mg protein−1 (n = 3). Bidirectional transport experiments across IPEC-J2 MDR1 monolayers showed that eletriptan is transported by P-gp, thus indicating that eletriptan is both a substrate of the H+/OC antiporter and P-gp. This was further confirmed in vivo, where the unbound brain-to-unbound plasma concentration ratio (Kp,uu) was 0.04 in wild type mice while the ratio rose to 1.32 in mdr1a/1b knockout mice. Conclusions We have demonstrated that the triptan family of compounds possesses affinity for the H+/OC antiporter proposing that the putative H+/OC antiporter plays a role in the BBB transport of triptans, particularly eletriptan. Our in vivo studies indicate that eletriptan is subjected to simultaneous brain uptake and efflux, possibly facilitated by the putative H+/OC antiporter and P-gp, respectively. Our findings offer novel insights into the potential central site of action involved in migraine treatment with triptans and highlight the significance of potential transporter related drug-drug interactions. Graphical Abstract

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Section: Methodsmentioning

confidence: 99%

The putative proton-coupled organic cation antiporter is involved in uptake of triptans into human brain capillary endothelial cells

Svane,

Pedersen,

Rodenberg

et al. 2024

Fluids Barriers CNS

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“…Langthaler et al [ 78 ] have also reported in their recent study that the most prominent difference in Kp,uu across different species (mini pig, dog, monkey, human) was observed for transporter substrates. Rodent transporter knockout models have also been employed to assess brain penetration in drug discovery [ 92 ]. P-gp is widely expressed at the BBB.…”

Section: Active Transport Across the Bbb (Efflux Transporters Influx ...mentioning

confidence: 99%

Experimental and Computational Methods to Assess Central Nervous System Penetration of Small Molecules

Gupta,

Feng,

Bhisetti

2024

Molecules

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In CNS drug discovery, the estimation of brain exposure to lead compounds is critical for their optimization. Compounds need to cross the blood–brain barrier (BBB) to reach the pharmacological targets in the CNS. The BBB is a complex system involving passive and active mechanisms of transport and efflux transporters such as P-glycoproteins (P-gp) and breast cancer resistance protein (BCRP), which play an essential role in CNS penetration of small molecules. Several in vivo, in vitro, and in silico methods are available to estimate human brain penetration. Preclinical species are used as in vivo models to understand unbound brain exposure by deriving the Kp,uu parameter and the brain/plasma ratio of exposure corrected with the plasma and brain free fraction. The MDCK-mdr1 (Madin Darby canine kidney cells transfected with the MDR1 gene encoding for the human P-gp) assay is the commonly used in vitro assay to estimate compound permeability and human efflux. The in silico methods to predict brain exposure, such as CNS MPO, CNS BBB scores, and various machine learning models, help save costs and speed up compound discovery and optimization at all stages. These methods enable the screening of virtual compounds, building of a CNS penetrable compounds library, and optimization of lead molecules for CNS penetration. Therefore, it is crucial to understand the reliability and ability of these methods to predict CNS penetration. We review the in silico, in vitro, and in vivo data and their correlation with each other, as well as assess published experimental and computational approaches to predict the BBB penetrability of compounds.

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“…Terminal blood samples were collected from all animals. Brain samples were prepared by homogenization using ultrasonication as described previously (Eneberg et al, 2022) and analyzed together with the plasma samples using LC-MS/MS (40). The lower limit of quanti cation was determined to 1.0 ng/mL in plasma and 5 ng/g in brain tissue for both eletriptan and diphenhydramine.…”

Section: In Vivo Brain Distribution By Neuropharmacokinetic Assessmen...mentioning

confidence: 99%

The putative proton-coupled organic cation antiporter is involved in uptake of triptans into human brain capillary endothelial cells

Svane,

Pedersen,

Rodenberg

et al. 2024

Preprint

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View full text Add to dashboard Cite

Background Triptans are anti-migraine drugs with a potential central site of action. However, it is not known to what extent triptans cross the blood-brain barrier (BBB). The aim of this study was therefore to determine if triptans pass the brain capillary endothelium and investigate the possible underlying mechanisms with focus on the involvement of the putative proton-coupled organic cation (H+/OC) antiporter. Additionally, we evaluated whether triptans interacted with the efflux transporter, P-glycoprotein (P-gp). Methods We investigated the cellular uptake characteristics of the prototypical H+/OC antiporter substrates, pyrilamine and oxycodone, and seven different triptans in the human brain microvascular endothelial cell line, hCMEC/D3. Triptan interactions with P-gp were studied using the IPEC-J2 MDR1 cell line. Lastly, in vivo neuropharmacokinetic assessment of the unbound brain-to-plasma disposition of eletriptan was conducted in wild type and mdr1a/1b knockout mice. Results We demonstrated that most triptans were able to inhibit uptake of the H+/OC antiporter substrate, pyrilamine, with eletriptan emerging as the strongest inhibitor. Eletriptan, almotriptan, and sumatriptan exhibited a pH-dependent uptake into hCMEC/D3 cells. Eletriptan demonstrated saturable uptake kinetics with an apparent Km of 89 ± 38 µM and a Jmax of 2.2 ± 0.7 nmol·min− 1·mg protein− 1 (n = 3). Bidirectional transport experiments across IPEC-J2 MDR1 monolayers showed that eletriptan is transported by P-gp, thus indicating that eletriptan is both a substrate of the H+/OC antiporter and P-gp. This was further confirmed in vivo, where the unbound brain-to-unbound plasma concentration ratio (Kp,uu) was 0.04 in wild type mice while the ratio rose to 1.32 in mdr1a/1b knockout mice. Conclusions We have demonstrated that the triptan family of compounds possesses affinity for the H+/OC antiporter proposing that the putative H+/OC antiporter plays a role in the BBB transport of triptans, particularly eletriptan. Our in vivo studies indicate that eletriptan is subjected to simultaneous brain uptake and efflux, possibly facilitated by the putative H+/OC antiporter and P-gp, respectively. Our findings offer novel insights into the potential central site of action involved in migraine treatment with triptans and highlight the significance of potential transporter related drug-drug interactions.

show abstract

Practical Application of Rodent Transporter Knockout Models to Assess Brain Penetration in Drug Discovery

Cited by 4 publications

References 42 publications

The putative proton-coupled organic cation antiporter is involved in uptake of triptans into human brain capillary endothelial cells

The putative proton-coupled organic cation antiporter is involved in uptake of triptans into human brain capillary endothelial cells

Experimental and Computational Methods to Assess Central Nervous System Penetration of Small Molecules

The putative proton-coupled organic cation antiporter is involved in uptake of triptans into human brain capillary endothelial cells

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